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Biomedical subjects

J Draper

Publications and source records attributed to J Draper.

At least 37 records · Page 2Linked to original sources
PubMed1994

The developmental expression of the asparagus intracellular PR protein (AoPR1) gene correlates with sites of phenylpropanoid biosynthesis.

Previous reports have described the induction, by either wounding or attempted pathogen invasion, of an Asparagus officinalis intracellular pathogenesis-related (AoPR1) promoter-GUS gene fusion in transgenic tobacco. Here we describe the unexpected developmental expression pattern of the AoPR1-GUS gene which correlates well, temporally and spatially, with the developmental expression observed for GUS fusions with promoters derived from genes coding for enzymes in the 'core phenylpropanoid pathway'. Analysis of endogenous AoPR1 gene expression in asparagus and both AoPR1-GUS and AoPR1-luciferase gene fusions in transgenic tobacco suggests that the AoPR1 promoter directs similar cell-specific transcription patterns in both asparagus and transgenic tobacco. The AoPR1 promoter contains sequence motifs similar to those implicated as important in the regulation of phenylpropanoid pathway genes and another 'intracellular' PR gene. Treatment with salicylic acid enhances AoPR1 promoter gene activity both in tobacco and in asparagus.

Base Sequence↗
PubMed1993

Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures.

A synthetic gene encoding an anti-phytochrome single-chain Fv (scFv) antibody bearing an N-terminal signal peptide has been used to transform tobacco plants. Immunoblot analysis showed that transformed plants accumulate high levels of scFv protein, accounting for up to 0.5% of the total soluble protein fraction, which could be extracted by simple infiltration and centrifugation of leaf tissue. A substantial proportion of the scFv protein extracted in this way was found to possess antigen-binding activity. Callus cell suspension cultures derived from transformed plants secrete functional scFv protein into the surrounding medium. Compared with the levels of scFv protein observed in plants expressing the native scFv gene, the incorporation of an N-terminal signal peptide, to target the scFv to the apoplast, results in elevated accumulation of the protein.

Amino Acid Sequence↗
PubMed1993

A wound-induced promoter driving npt-II expression limited to dedifferentiated cells at wound sites is sufficient to allow selection of transgenic shoots.

There is much data to indicate that only a small number of cells in plant explants are competent for stable transformation by Agrobacterium. Circumstantial evidence suggests that certain cells reentering cell division at wound sites are competent for transformation by Agrobacterium. We have discovered a member of the intracellular PR gene family from asparagus (AoPR1) which is strongly expressed upon wounding and during the reactivation of the cell cycle in cultured asparagus cells, but which shows very little expression in intact plant tissues. The promoter from the AoPR1 gene was fused to an intron-containing GUS reporter gene and shown to be more strongly expressed than the commonly used CaMV 35S constitutive promoter in target cells for plant transformation. A transcriptional fusion of the AoPR1 promoter with an NPT-II gene was found to be a very efficient marker for the selection of transgenic tobacco callus. Expression of the AoPR1-NPT-II gene allowed efficient shoot formation on transgenic callus and efficient adventitious root formation on transgenic shoots. These latter observations provided firm evidence that transformation selection marker gene expression is most crucial at the early stages of the transformation process, during the establishment of transformed micro-calli.

Agrobacterium tumefaciens↗
PubMed1993

Isolation of an asparagus intracellular PR gene (AoPR1) wound-responsive promoter by the inverse polymerase chain reaction and its characterization in transgenic tobacco.

The Asparagus officinalis intracellular PR1 (AoPR1) gene is expressed in response to wounding and pathogen attack. We utilized the inverse polymerase chain reaction (IPCR) to isolate the cis-acting regulatory sequences of the AoPR1 gene following unsuccessful attempts to identify hybridizing clones in genomic libraries. Sequence analysis of two IPCR products revealed that a 347 bp intron was present in the AoPR1 gene and that it was probable that the AoPR1 regulatory sequence had been amplified. To test the AoPR1 cis-acting sequences for biological function a translational fusion was constructed with the beta-glucuronidase (GUS) reporter gene and tested in tobacco. These data demonstrated that sequences 982 bp from the probable start of transcription are sufficient to direct wound-inducible transcription and that there is no signal peptide encoded by the first 31 residues of the predicted AoPR1 protein. Histochemical localization of GUS activity in transgenic tobacco demonstrated strong activity localized to wound and pathogen invasion sites. GUS activity was also found in mature pollen grains.

Amino Acid Sequence↗
PubMed1993

Characterization of a new class of oleosins suggests a male gametophyte-specific lipid storage pathway.

The expression and sequences of two related antherspecific cDNAs (I3 and C98) of Brassica napus (oilseed rape) were examined. These cDNAs were found to exhibit significant predicted amino acid sequence similarity with a group of seed-specific proteins, the oleosins, which are involved in oil body membrane structure. Pollen of B. napus also contains oil bodies, and the synthesis and accumulation of these organelles correlates with expression of the I3 and C98 transcripts. The protein content of purified pollen oil bodies was therefore examined in order to determine the presence or absence of possible oleosins. One major protein species of 14 kDa was identified and subjected to N-terminal amino acid sequence determination. The sequence of this pollen oil body associated protein is homologous to the predicted sequence of one of the anther-specific cDNs, I3, implying that this cDNA represents an anther-specific oleosin gene expressed in developing pollen. The expression of other genes active during seed oil body formation was also examined, and these were found to be specific to the sporophyte, inferring the existence of specific components of the pathway of lipid synthesis and storage in the male gametophyte. These possibilities and the relationship between different oleosin genes are discussed.

Amino Acid Sequence↗
PubMed1993

Gametophytic and sporophytic expression of an anther-specific Arabidopsis thaliana gene.

Genomic and cDNA clones of the anther-specific APG gene from Arabidopsis thaliana and Brassica napus, which encodes a novel proline-rich protein, were isolated and characterized. Southern blotting and Northern analysis of male fertile and cytoplasmic male sterile varieties of B. napus showed that the APG gene is present as a single copy in the Arabidopsis genome, and that the B. napus APG gene is a member of a small anther-specific gene family. Analysis of developmentally staged B. napus flower buds indicated that APG transcript is confined to the anther during the period of microspore development. Reporter gene fusions established that the APG promoter directs expression in a number of cell types in anthers of transformed plants. This expression is consistent with the temporal pattern of mRNA accumulation in B. napus buds and follows a complex developmental pattern. Most significantly, the promoter is active in both sporophytic and gametophytic cell types, with activity of the transgene in each cell type being delineated by various cytological markers.

Amino Acid Sequence↗
PubMed1992

Characterisation of a wound-induced transcript from the monocot asparagus that shares similarity with a class of intracellular pathogenesis-related (PR) proteins.

We report the isolation and characterisation of a wound-induced cDNA designated AoPR1 from a suspension of mesophyll cells that had been mechanically isolated from cladodes of light-grown Asparagus officinalis seedlings by grinding in a mortar and pestle. The transcript abundance is up-regulated following cell separation and in chopped mesocotyl tissue from dark-grown seedlings. The expression of AoPR1 was shown by northern analysis to be located around the site of damage. Sequence analysis revealed similarity between the predicted AoPR1 polypeptide and bean PvPR1 and PvPR2 proteins, the potato pSTH2 protein, the pea PI49 protein, the parsley PcPR1-1 protein and a major pollen allergen from birch (BetvI). These transcripts have been shown to be induced in response to microbial attack or fungal elicitation. To our knowledge, this is the first example of a monocot cDNA belonging to this class of intracellular pathogenesis-related proteins (IPRs).

Amino Acid Sequence↗
PubMed1992

The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene.

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene beta-glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase. The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and alpha-amylase inhibitors.

Amino Acid Sequence↗
PubMed1992

Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco.

Male sterility in a petunia cytoplasmic male sterile line has been attributed to the early appearance of active callase, a beta-1,3-glucanase, in the anther locule. This leads to premature dissolution of the callose walls surrounding the microsporogenous cells. We have mimicked this aspect of the petunia line in transgenic tobacco by engineering the secretion of a modified pathogenesis-related vacuolar beta-1,3-glucanase from the tapetum prior to the appearance of callase activity in the locule. Plants expressing the modified glucanase from tapetum-specific promoters exhibited reduced male fertility, ranging from complete to partial male sterility. Callose appearance and distribution are normal in the male sterile transgenic plants up to prophase I, whereupon callose is prematurely degraded. Meiosis and cell division occur normally. The resultant microspores have an abnormally thin cell wall that lacks sculpturing. The tapetum shows hypertrophy. Male sterility is probably caused by bursting of the aberrant microspores at a time corresponding to microspore release. These results demonstrate that premature callose degradation is sufficient to cause male sterility and suggest that callose is essential for the formation of a normal microspore cell wall.

Base Sequence↗
PubMed1992

Expression of Helenium virus S coat protein in Escherichia coli, in vitro in rabbit reticulocyte lysate and transgenic tobacco.

The coat protein open reading frame (ORF) sequence of Helenium virus S (HelVS) was cloned and expressed in E. coli, rabbit reticulocyte and transgenic tobacco. In E. coli the size of the protein was identical to that obtained for the coat protein from purified virus particles and less than that predicted for the fusion protein. This may be due to ribosome binding at a potential ribosome binding site present on the viral sequence, approximately 45 nucleotides upstream from the initiating methionine of the coat protein ORF. This region of HelVS, equivalent to the 1.5 kb subgenomic RNA, also produced high levels of protein when transcribed and translated in vitro. When introduced into Nicotiana tabacum by leaf disk transformation via Agrobacterium tumefaciens, high levels of stable coat protein were detected which were identical in molecular weight to that of HelVS coat protein and constituted approximately 0.1-0.5% of the total extracted protein.

Animals↗
PubMed1991

Patterns of gene expression in developing anthers of Brassica napus.

The relationship between bud length, anther length and stage of anther development has been investigated in Brassica napus using a series of cytological markers that define steps in the process of male gametogenesis. It was determined that bud length is directly related to anther length and that anther or bud length is tightly linked to the stage of male gametogenesis within the anther. This simple correlation has enabled the construction of cDNA libraries representing transcripts expressed in defined stages of anther development, and the detailed examination of the developmental pattern of expression of anther RNAs. Two anther cDNA libraries were constructed, one from anthers of 1.2-1.8 mm long buds (sporogenesis library) and one from anthers of 1.8-4.0 mm long buds (microspore development library). A total of 19 independent cDNAs have been isolated by differential screening whose temporal expression patterns overlap and which together cover the stages of anther development from pre-meiotic microsporocytes to tri-nucleate pollen grains. The pattern of expression of each of these clones is unique and indicates that stages of anther development which cannot be easily distinguished by light microscopy can be recognised by virtue of the absence or presence of certain RNAs. Three cDNAs isolated from the sporogenesis library have been shown by in situ hybridisation to be tapetum-specific. In contrast, five clones isolated from the microspore development library are microspore-specific. These clones exhibit a pattern of expression different to those previously described in that their transcripts are absent in mature pollen grains. Thus these RNAs are probably required in microspore development rather than for the growth of the germinating pollen grain.

Blotting, Northern↗
PubMed1991

A Brassica napus mRNA expressed specifically in developing microspores.

The I3 cDNA isolated from a library made from 2-4 mm (immature) anthers of Brassica napus shows microspore-specific expression. Homologous transcripts are detected in buds and anthers of male-fertile plants, but not in green tissues, roots, or in cytoplasmic male-sterile buds. High expression of the transcript is limited to microspores entering and undergoing mitosis. The predicted peptide sequence of the cDNA shows an unusual repeated alanine/proline motif at the C-terminus, which may be of importance in the native protein structure.

Amino Acid Sequence↗
PubMed1990

Perimenopausal women's views on taking hormone replacement therapy to prevent osteoporosis.

OBJECTIVE: To find out perimenopausal women's views on taking hormone replacement therapy to prevent osteoporosis. DESIGN: Semistructured postal questionnaire survey; the questionnaire was enclosed with a letter to explain the purpose of the study and to give patients some information about hormone replacement therapy and osteoporosis. SETTING: Mixed social class, urban general practice with 12,000 registered patients. PATIENTS: All women aged 50, 51, or 52 were selected from the age-sex register; 102 women were identified and 84 (87%) replied to the questionnaire. MAIN OUTCOME MEASURES: Response rates to questions on interest in taking hormone replacement therapy, the importance of preventing osteoporosis, and worries about side effects of the treatment. RESULTS: More than three quarters of the respondents (65) were interested in taking hormone replacement therapy to prevent osteoporosis, but 48 of them would have liked further information. Sixty four women thought that it was very important to prevent osteoporosis. More than half of the respondents (51) were worried about side effects of the treatment, but only 15 women thought that continued menstruation would be a major disadvantage. CONCLUSIONS: There is considerable interest among perimenopausal women in taking hormone replacement therapy to prevent osteoporosis. The resource implications for primary care in meeting this interest are substantial.

Attitude to Health↗
PubMed1990

Superior oblique palsy: diagnosis and treatment.

To pinpoint the diagnostic dilemmas and to look for differentiating preoperative features in cases of masked bilateral superior oblique palsy, the authors reviewed the charts of 50 patients (26 males and 24 females with an average age at presentation of 25.6 years) with an established diagnosis of superior oblique palsy seen between 1980 and 1987. Most patients had a history of symptoms from childhood or following trauma. Typically, patients complained of torticollis and diplopia and manifested inferior oblique overaction and superior oblique underaction of the paretic eye. Twenty-two of the patients underwent a total of 28 operations; surgery resulted in elimination of symptoms and neutralization of the hyperdeviation in 77%. On the basis of this experience the authors discuss the presenting features and the differential diagnosis of superior oblique palsy and present a rational approach to surgical intervention.

Adolescent↗
PubMed1989

Bilateral congenital third cranial nerve palsy.

We describe a patient in whom bilateral congenital third cranial nerve palsy was diagnosed at 2 weeks of age. The ptosis was treated at 3 weeks with bilateral eyelid suspension surgery, and surgical repair of the exotropia was done at 4 months. To our knowledge this is the fourth reported case of this condition. It was probably caused by a single lesion involving the oculomotor and possibly the trochlear nuclei in the brain stem. Magnetic resonance imaging scans supported this hypothesis and suggested other central nervous system anomalies.

Child, Preschool↗
PubMed1987

Evaluation of an integrated community antenatal clinic.

The obstetric outcome and experience of care of 96 pregnant women attending an integrated community antenatal clinic staffed by general practitioners, a community midwife and an obstetric accredited senior registrar were compared with those of 100 women receiving traditional shared antenatal care. The views of the women and their practitioners were sought; obstetric data were obtained from obstetric notes, hospital records and cooperation cards.Fewer women attending the community clinic suffered from hypertension than women receiving shared care. The women attending the clinic reported that it had a friendly, relaxed and personal atmosphere. They also reported less inconvenience and a shorter waiting time for the obstetrician than women receiving shared care. They received greater continuity of care from the obstetrician but less from the general practitioners and community midwives than the control women. There was greater satisfaction with communication with staff among women attending the clinic, with the exception of the midwife whose role was not sufficiently well delineated. Practitioners in the integrated scheme appreciated the close working arrangements but experienced an increase in administrative tasks.

Community Health Services↗